EP4424698A2 - Verfahren und zusammensetzungen zur proteinsynthese und -sekretation - Google Patents

Verfahren und zusammensetzungen zur proteinsynthese und -sekretation Download PDF

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EP4424698A2
EP4424698A2 EP24185760.6A EP24185760A EP4424698A2 EP 4424698 A2 EP4424698 A2 EP 4424698A2 EP 24185760 A EP24185760 A EP 24185760A EP 4424698 A2 EP4424698 A2 EP 4424698A2
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Prior art keywords
protein
nucleic acid
cell
sequence
seq
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English (en)
French (fr)
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EP4424698A3 (de
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Pamela BOTERO BESADA-LOMBANA
Laura KATZ
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Helaina Inc
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Helaina Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A23C9/1526—Amino acids; Peptides; Protein hydrolysates; Nucleic acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • aspects of this invention relate to at least the fields of microbiology, genetics, and biotechnology.
  • Yeast is a desirable host for production of recombinant proteins due to its rapid growth, its ability to reach high cell densities, to grow on defined minimal media, achieve high protein yields and conduct eukaryotic post-translational modifications.
  • the most relevant yeast for protein production is Pichia pastoris ( Komagataella pastoris, Komagataella phaffii ) due to the wide availability of genomic information and molecular tools for genomic manipulation. These have enabled the use of Pichia pastoris for production of GRAS ingredients based on the FDA criteria.
  • Pichia pastoris is capable of secreting active recombinant proteins, while maintaining low-level secretion of endogenous proteins.
  • secreted proteins are first targeted from the cytoplasm to the lumen endoplasmic reticulum (ER) via translocation.
  • Translocation into the ER can take place either post-translationally (i.e., once the polypeptide chain has been synthesized) or co-translationally (i.e., during mRNA translation into its amino acid sequence).
  • Post-translational translocation requires chaperones that maintain the polypeptide chain in a loose conformation in the cytosol as well as the action of the ER-resident chaperone Kar2, which acts as a molecular ratchet. Consequently, this process can be hindered by partially folded domains and/or cytosolic aggregation.
  • proteins are glycosylated, their disulfide bonds are isomerized, and they fold to their native state. Proteins that are successfully folded then transit to the Golgi complex, where further glycosylation takes place before being packed into secretory granules that fuse to the cell membrane, releasing the protein to the extracellular milieu.
  • the most widely used in Pichia pastoris is the leader peptide of the mating factor alpha of S. cerevisiae. It is comprised of two distinct regions: ii) the first 19 amino acid pre-region that promotes post-translational translocation and is cleaved upon ER entry ii) a 70 amino acid pro-segment that serves as an ER-to-Golgi export signal and it is cleaved in the Golgi Apparatus at the dibasic amino acid cleavage site KR.
  • aspects of the present disclosure address certain needs by providing novel secretion signal peptides effective in improving extracellular production of proteins, including mammalian proteins such as, for example, human milk proteins. Certain aspects of the disclosure are based, at least in part, on the development of signal peptides generated from the in-frame fusion of 1) pre-secretion peptides of P. pastoris from either i) the alpha subunit of the oligosaccharyltransferase complex of the ER lumen (Ost1) or ii) the GPI-anchored protein Pst1 with 2) the pro-region of either i) the S. cerevisiae mating factor or ii) pro-region of P. pastoris Epxl.
  • nucleic acids encoding such secretion signal peptides, in some cases linked to a recombinant protein such as a human milk protein, as well as cells comprising such nucleic acids and methods for producing and collecting recombinant proteins from such cells.
  • the sequence comprises SEQ ID NO:1, 2, 3, or 4.
  • the polypeptide further comprises a sequence of a mammalian protein.
  • the mammalian protein is a human milk protein.
  • the human milk protein is secretory IgA (sIgA), xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, ⁇ -casein, ⁇ -casein, leptin, lysozyme, or ⁇ -lactalbumin.
  • the human milk protein is human lactoferrin.
  • the sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1.
  • the sequence comprises SEQ ID NO:1.
  • the isolated nucleic acid comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:41.
  • the nucleic acid sequence comprises SEQ ID NO:41.
  • the polypeptide comprises SEQ ID NO:5.
  • the isolated nucleic acid comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:46.
  • the nucleic acid sequence comprises SEQ ID NO:46.
  • the sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2.
  • the sequence comprises SEQ ID NO:2.
  • the isolated nucleic acid comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:42.
  • the nucleic acid sequence comprises SEQ ID NO:42.
  • the polypeptide comprises SEQ ID NO:6.
  • the isolated nucleic acid comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:47.
  • the nucleic acid sequence comprises SEQ ID NO:47.
  • the sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3.
  • the sequence comprises SEQ ID NO:3.
  • the isolated nucleic acid comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:43.
  • the nucleic acid sequence comprises SEQ ID NO:43.
  • the polypeptide comprises SEQ ID NO:7.
  • the isolated nucleic acid comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:48.
  • the nucleic acid sequence comprises SEQ ID NO:48.
  • the sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:4.
  • the sequence comprises SEQ ID NO:4.
  • the isolated nucleic acid comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:44.
  • the nucleic acid sequence comprises SEQ ID NO:44.
  • the polypeptide comprises SEQ ID NO:8.
  • the isolated nucleic acid comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:49.
  • the nucleic acid sequence comprises SEQ ID NO:49.
  • nucleic acid e.g., an isolated nucleic acid or sequence or portion thereof
  • the cell is a fungal cell.
  • the fungal cell is a Arxula, Aspegillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodamaea, Komagataella, Leucosporidiella, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickerhamomyces, or Yarrowia cell.
  • the cell is a yeast cell. In some embodiments, the yeast cell is a Komagataella cell. In some embodiments, the yeast cell is a Komagataella phaffii, Komagataella pastoris, or Komagataella pseudopastoris cell. In some aspects, the nucleic acid is integrated into the genome of the cell. In some aspects, the nucleic acid is not integrated into the genome of the cell.
  • a method for producing a secreted protein comprising growing an engineered eukaryotic cell of the present disclosure under conditions sufficient to secrete the polypeptide from the cell.
  • the method further comprises collecting the secreted protein.
  • the secreted protein is a human milk protein.
  • the human milk protein is secretory IgA (sIgA), xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, ⁇ -casein, ⁇ -casein, leptin, lysozyme, or ⁇ -lactalbumin.
  • the human milk protein is human lactoferrin. In some embodiments, the human milk protein comprises one or more human-like N-glycans. In some embodiments, the method further comprises generating a mixture comprising the human milk protein and one or more components of an infant formula.
  • an engineered yeast cell comprising a nucleic acid encoding a polypeptide comprising a sequence having at least 90% sequence identity to SEQ ID NO:1, 2, 3, or 4.
  • the sequence comprises SEQ ID NO:1, 2, 3, or 4.
  • the sequence comprises SEQ ID NO:1.
  • the sequence comprises SEQ ID NO:2.
  • the sequence comprises SEQ ID NO:3.
  • the sequence comprises SEQ ID NO:4.
  • the polypeptide further comprises a sequence of a mammalian protein.
  • the mammalian protein is a human milk protein.
  • the human milk protein is secretory IgA (sIgA), xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, ⁇ -casein, ⁇ -casein, leptin, lysozyme, or ⁇ -lactalbumin.
  • the human milk protein is human lactoferrin.
  • an engineered yeast cell comprising: (a) a first nucleic acid encoding a polypeptide comprising: (i) a sequence having at least 90% sequence identity to SEQ ID NO:1, 2, 3, or 4 and (ii) a sequence of a human milk protein; and (b) a second nucleic acid encoding an alpha-1,2-mannosidase (Man-I) protein, wherein the cell does not express a functional OCH1 protein.
  • the sequence of (i) comprises SEQ ID NO:1, 2, 3, or 4.
  • the human milk protein is secretory IgA (sIgA), xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, ⁇ -casein, ⁇ -casein, leptin, lysozyme, or ⁇ -lactalbumin.
  • the human milk protein is human lactoferrin.
  • the human milk protein is human ⁇ -lactalbumin.
  • the Man-I protein is fused to a HDEL C-terminal tag.
  • the cell further comprises a third nucleic acid encoding one or more of: (a) a N-acetylglucosaminyltransferase I (GnT-I) protein; (b) an ⁇ -1,3/6-Mannosidase (Man-II) protein; (c) a ⁇ -1,2-acetylglucosaminyltransferase (GnT-II) protein; and (d) a ⁇ -1,4-galactosyltransferase (GalT) protein.
  • the yeast cell is a Komagataella cell.
  • the yeast cell is a Komagataella phaffii, Komagataella pastoris, or Komagataella pseudopastoris cell.
  • the nucleic acid is integrated into the genome of the cell. In some aspects, the nucleic acid is not integrated into the genome of the cell.
  • Described herein is the generation of novel synthetic secretion signal peptides.
  • cells e.g., fungal cells such as yeast cells
  • exogenous proteins e.g., human milk proteins
  • the in-frame fusion of "pre-region" sequences from P. pastoris Ost1 or Pst1 and "pro-region” sequences from S. cerevisiae mating factor ⁇ or P. pastoris Epxl can facilitate increased extracellular protein production compared with previously used signal peptides.
  • the disclosed signal peptides include, for example, peptides comprising SEQ ID NOs:1, 2, 3, or 4, as well as peptides comprising 1, 2, 3, 4, or 5 amino acid substitutions (or more) relative to SEQ ID NO: 1, 2, 3, or 4.
  • in-frame fusion of these hybrid signal peptides to the N-terminus of mammalian proteins promotes highly efficient protein secretion.
  • biologically-active portion refers to an amino acid sequence that is less than a full-length amino acid sequence, but exhibits at least one activity of the full length sequence.
  • a biologically-active portion of an enzyme may refer to one or more domains of an enzyme having the catalytic activity of the enzyme (i.e., may be a catalytic domain).
  • a biologically-active portion of an enzyme is a portion of the enzyme comprising a catalytic domain of the enzyme.
  • Bioly-active portions of a protein include peptides or polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the protein, which include fewer amino acids than the full length protein, and exhibit at least one activity (e.g., enzymatic activity, functional activity, etc.) of the protein.
  • exogenous refers to anything that is introduced into a cell or has been introduced into a cell.
  • An "exogenous nucleic acid” is a nucleic acid that enters or has entered a cell through the cell membrane.
  • An “exogenous nucleic acid sequence” is a nucleic acid sequence of an exogenous nucleic acid.
  • An exogenous nucleic acid may contain a nucleotide sequence that exists in the native genome of a cell and/or nucleotide sequences that did not previously exist in the cell's genome.
  • Exogenous nucleic acids include exogenous genes.
  • exogenous gene is a nucleic acid that codes for the expression of an RNA and/or protein that has been introduced into a cell (e.g., by transformation/transfection), and is also referred to as a "transgene.”
  • a cell comprising an exogenous nucleic acid may be referred to as a recombinant cell, into which additional exogenous gene(s) may be introduced.
  • the exogenous gene may be from the same or different species relative to the cell being transformed.
  • an exogenous gene can include a native gene that occupies a different location in the genome of the cell or is under different control, relative to the endogenous copy of the gene.
  • An exogenous gene may be present in more than one copy in the cell.
  • An exogenous gene may be maintained in a cell as an insertion into the genome (nuclear, mitochondrial, or plastid) or as an episomal molecule.
  • operable linkage refers to a functional linkage between two nucleic acid sequences, such a control sequence (typically a promoter) and the linked sequence (typically a sequence that encodes a protein, also called a coding sequence).
  • a promoter is in operable linkage with a gene if it can mediate transcription of the gene.
  • mutant refers to the composition of a cell or parent cell prior to a transformation event.
  • a “native gene” also “endogenous gene” refers to a nucleotide sequence that encodes a protein that has not been introduced into a cell by a transformation event.
  • a “native protein” also “endogenous protein” refers to an amino acid sequence that is encoded by a native gene.
  • Recombinant refers to a cell, nucleic acid, protein, or vector, which has been modified due to introduction of an exogenous nucleic acid or alteration of a native nucleic acid. Resulting cells, nucleic acids, proteins or vectors are considered recombinant, as are progeny, offspring, duplications or replications of these are also considered recombinant. Thus, e.g., recombinant cells can express genes that are not found within the native (non-recombinant) form of the cell or express native genes differently than those same genes are expressed by a non-recombinant cell.
  • Recombinant cells can, without limitation, include recombinant nucleic acids that encode for a gene product or for suppression elements such as mutations, knockouts, antisense, interfering RNA (RNAi), or dsRNA that reduce the levels of active gene product in a cell.
  • a "recombinant nucleic acid” is derived from nucleic acid originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases, ligases, exonucleases, and endonucleases, or otherwise is in a form not normally found in nature.
  • a recombinant nucleic acid refers to nucleotide sequences that comprise an endogenous nucleotide sequence and an exogenous nucleotide sequence; thus, an endogenous gene that has undergone recombination with an exogenous promoter is a recombinant nucleic acid.
  • a "recombinant protein” is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid.
  • Transformation refers to the transfer of a nucleic acid into a host organism or the genome of a host organism.
  • Host organisms (and their progeny) containing the transformed nucleic acid fragments are referred to as “recombinant”, “transgenic” or “transformed” organisms.
  • isolated polynucleotides of the present disclosure can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell.
  • Such a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell.
  • expression vectors include, for example, one or more cloned genes under the transcriptional control of 5' and 3' regulatory sequences and a selectable marker.
  • Such vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or location-specific expression), a transcription initiation start site, a ribosome binding site, a transcription termination site, and/or a polyadenylation signal.
  • a cell may be transformed with a single genetic element, such as a promoter, which may result in genetically stable inheritance upon integrating into the host organism's genome, such as by homologous recombination.
  • transformed cell refers to a cell that has undergone a transformation.
  • a transformed cell comprises the parent's genome and an inheritable genetic modification.
  • Embodiments include progeny and offspring of such transformed cells.
  • vector refers to the means by which a nucleic acid can be propagated and/or transferred between organisms, cells, or cellular components.
  • Vectors include plasmids, linear DNA fragments, viruses, bacteriophage, pro-viruses, phagemids, transposons, and artificial chromosomes, and the like, that may or may not be able to replicate autonomously or integrate into a chromosome of a host cell.
  • “Individual,” “subject,” and “patient” are used interchangeably and can refer to a human or non-human.
  • A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.
  • A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.
  • “and/or” operates as an inclusive or.
  • compositions and methods for their use can "comprise,” “consist essentially of,” or “consist of” any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of” any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed embodiment.
  • a "protein” or “polypeptide” refers to a molecule comprising at least five amino acid residues.
  • wild-type refers to the endogenous version of a molecule that occurs naturally in an organism.
  • wild-type versions of a protein or polypeptide are employed, however, in many embodiments of the disclosure, a modified protein or polypeptide is employed.
  • a “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide.
  • a modified/variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified/variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects.
  • a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein or, optionally, a protein in which any signal sequence has been removed.
  • the protein may be isolated directly from the organism of which it is native, produced by recombinant DNA/exogenous expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods.
  • SPPS solid-phase peptide synthesis
  • recombinant may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.
  • the size of a protein or polypeptide may comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,
  • polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.).
  • domain refers to any distinct functional or structural unit of a protein or polypeptide, and generally refers to a sequence of amino acids with a structure or function recognizable by one skilled in the art.
  • polynucleotide refers to a nucleic acid molecule that either is recombinant or has been isolated from total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (nucleic acids 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences.
  • Polynucleotides may be single- stranded (coding or antisense) or double- stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or noncoding sequences may, but need not, be present within a polynucleotide.
  • nucleic acid refers to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization).
  • this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants.
  • a nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein.
  • polynucleotide variants having substantial identity to the sequences disclosed herein; those comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, including all values and ranges there between, compared to a polynucleotide sequence provided herein using the methods described herein (e.g., BLAST analysis using standard parameters).
  • the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 90%, and in some cases 95% and above, identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide.
  • nucleic acid segments may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably.
  • the nucleic acids can be any length. They can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides in length, and/or can comprise one or more additional sequences, for example, regulatory sequences, and/or be a part of a larger nucleic acid, for example, a vector.
  • nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol.
  • a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy.
  • a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.
  • polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins of the disclosure may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) or more variant amino acids or nucleic acid substitutions or be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein)
  • the protein or polypeptide may comprise amino acids 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114
  • the protein, polypeptide, or nucleic acid may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113
  • the polypeptide, protein, or nucleic acid may comprise at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110
  • nucleic acid molecule or polypeptide starting at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,
  • nucleotide as well as the protein, polypeptide, and peptide sequences for various genes have been previously disclosed, and may be found in the recognized computerized databases.
  • Two commonly used databases are the National Center for Biotechnology Information's Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov/) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org).
  • the coding regions for these genes may be amplified and/or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.
  • compositions of the disclosure there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and/or protein per ml.
  • concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or any range derivable therein).
  • EC Enzyme Classification
  • aspects of the present disclosure are directed to synthetic signal peptides, and polynucleotides and nucleic acids encoding such signal peptides. Also disclosed are cells comprising such signal peptides, and methods for using cells in production and secretion of a protein (e.g., mammalian protein such as human milk protein).
  • a protein e.g., mammalian protein such as human milk protein.
  • signal peptide (or “signal peptide sequence”) describes any peptide able to, when present at the N-terminal end of a newly synthesized polypeptide, direct the polypeptide across or into a cell membrane of a cell (e.g., the plasma membrane, the endoplasmic reticulum membrane, etc.).
  • a signal peptide of the present disclosure is able to direct a polypeptide into a cell's secretory pathway and subsequent secretion of the polypeptide (described herein as a "secretion signal peptide").
  • aspects of the disclosure relate to synthetic signal peptides comprising:
  • polypeptides comprising a signal peptide of the present disclosure.
  • nucleic acids encoding such polypeptides.
  • cells expressing polypeptides comprising a signal peptide of the present disclosure.
  • a polypeptide of the present disclosure comprises SEQ ID NO:1. In some embodiments, a polypeptide of the present disclosure comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1. In some aspects, a polypeptide of the present disclosure comprises a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions (or more) relative to SEQ ID NO:1.
  • a polypeptide of the present disclosure comprises SEQ ID NO:2. In some embodiments, a polypeptide of the present disclosure comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:2. In some aspects, a polypeptide of the present disclosure comprises a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions (or more) relative to SEQ ID NO:2.
  • a polypeptide of the present disclosure comprises SEQ ID NO:3. In some embodiments, a polypeptide of the present disclosure comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:3. In some aspects, a polypeptide of the present disclosure comprises a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions (or more) relative to SEQ ID NO:3.
  • a polypeptide of the present disclosure comprises SEQ ID NO:4. In some embodiments, a polypeptide of the present disclosure comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:4. In some aspects, a polypeptide of the present disclosure comprises a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions (or more) relative to SEQ ID NO:4.
  • secretory proteins also “secreted proteins”
  • compositions comprising secretory proteins, methods of expressing secretory proteins, and methods of use thereof.
  • a secretory protein describes any protein secreted outside a cell.
  • a secretory protein of the disclosure is a protein present in a human secretion, such as, for example, colostrum, milk, tears, seminal fluid, vaginal fluid, saliva, or other secretion.
  • a secretory protein of the disclosure is a human milk protein.
  • a secretory protein of the disclosure is not a human milk protein.
  • aspects of the present disclosure include human milk proteins, as well as compositions (e.g., infant formula compositions) comprising human milk proteins, methods of producing human milk proteins, and methods of use thereof.
  • cells expressing a human milk protein linked to a signal peptide of the present disclosure e.g., comprising SEQ ID NOs: 1, 2, 3, or 4.
  • a "human milk protein” describes any protein present in human breast milk.
  • a human milk protein includes a protein derived from (e.g., isolated from) human breast milk, as well as any protein produced by other means (e.g., recombinant expression, chemical synthesis, etc.) having an amino acid sequence of a protein present in human breast milk.
  • Human milk proteins contemplated herein include, but are not limited to, secretory IgA (sIgA), human serum albumin, xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, ⁇ -casein, ⁇ -casein, leptin, lysozyme, and ⁇ -lactalbumin.
  • a human milk protein of the disclosure is a human whey protein.
  • a human milk protein of the disclosure is a recombinant human milk protein (e.g., produced by a non-mammalian cell such as a yeast cell).
  • Human-like glycans (also “human-like glycan structures") describe glycans having structures present in human glycoproteins.
  • Such glycans include, for example, hybrid N-glycans, complex N-glycans, bi-antennary, tri-antennary, and tetra-antennary N-glycans, and glycans comprising sialic acid, galactose, N-acetylgalactosamine, or fucose.
  • Human-like glycans include those having a Man3GlcNAc2 core structure.
  • human milk proteins of the disclosure include those having one or more human-like glycans, for example hybrid N-glycans, complex N-glycans, bi-antennary N-glycans, tri-antennary N-glycans, tetra-antennary N-glycans, and combinations thereof.
  • recombinant human milk proteins comprising one or more human-like glycans.
  • recombinant protein include, for example, those produced by engineered mammalian, fungal, yeast, bacterial, or other cells, including engineered cells described elsewhere herein.
  • recombinant proteins have a glycan pattern that different from a glycan pattern of a corresponding natural human milk protein.
  • a recombinant human lactoferrin comprising one or more human-like glycans, where the lactoferrin has a glycan pattern that is different from a glycan pattern of any naturally occurring human lactoferrin (e.g., human lactoferrin in human breast milk).
  • lactoferrin as well as compositions comprising lactoferrin, including infant formula compositions.
  • cells expressing human lactoferrin linked to a signal peptide of the present disclosure e.g., comprising SEQ ID NOs: 1, 2, 3, or 4.
  • Lactoferrin also "lactotransferrin" is a whey protein found in exocrine fluids such as breast milk and is encoded by the LTF gene. Without wishing to be bound by theory, lactoferrin is understood to have antimicrobial and anti-inflammatory properties.
  • Certain aspects of the disclosure are directed to human lactoferrin (UniProtKB/Swiss-Prot accession number P02788), including isoforms thereof.
  • the full sequence of human lactoferrin, including signal peptide, is provided as SEQ ID NO:34.
  • the sequence of mature human lactoferrin following cleavage of the signal peptide is provided as SEQ ID NO:9.
  • Table 2 - Human Lactoferrin sequences Protein Sequence SEQ ID NO Full length human lactoferrin 34 Mature human lactoferrin 9 SP1-human lactoferrin 5 SP2-human lactoferrin 6 SP3-human lactoferrin 7 SP4-human lactoferrin 8 P. pastoris codon-optimized Human lactoferrin gene 45 SP1-codon optimized hLF gene 46 SP2-codon optimized hLF gene 47 SP3-codon optimized hLF gene 48 SP4-codon optimized hLF gene 49
  • a human lactoferrin of the present disclosure is a recombinant human lactoferrin (rhLactoferrin).
  • a recombinant human lactoferrin of the disclosure is obtained from a mammalian, fungal, yeast, bacterial, or other cell.
  • a recombinant human lactoferrin of the disclosure is not obtained from a mammalian cell.
  • a recombinant human lactoferrin of the disclosure is obtained from a fungal cell.
  • the fungal cell may be, for example, a Arxula, Aspegillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodamaea, Komagataella, Leucosporidiella, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickerhamomyces, or Yarrowia cell.
  • the fungal cell is a yeast cell.
  • the yeast cell is yeast cell is a Komagataella cell (e.g., Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris ) . Additional cells suitable for recombinant protein production are recognized in the art and contemplated herein.
  • a recombinant human lactoferrin of the disclosure is obtained from a bacterial cell.
  • a human lactoferrin of the disclosure is isolated from a natural source.
  • human lactoferrin having at least one hybrid or complex N-glycan comprises a glycan comprising one or more of sialic acid, galactose, N-acetylgalactosamine, or fucose.
  • the human lactoferrin comprises a bi-antennary, tri-antennary, or tetra-antennary N-glycan.
  • human lactoferrin having one or more hybrid, complex, bi-antennary, tri-antennary, or tetra-antennary N-glycan may be useful in, for example, infant formula or other nutritional compositions or supplements.
  • aspects of the present disclosure are directed to alpha-lactalbumin, as well as compositions comprising alpha-lactalbumin, including infant formula compositions.
  • cells expressing human alpha-lactalbumin linked to a signal peptide of the present disclosure e.g., comprising SEQ ID NOs: 1, 2, 3, or 4.
  • Alpha-lactalbumin also " ⁇ -lactalbumin”
  • ⁇ -lactalbumin is a whey protein found in breast milk and is encoded by the LALBA gene.
  • Certain aspects of the disclosure are directed to human ⁇ -lactalbumin (UniProtKB/Swiss-Prot accession number P00709), including isoforms thereof.
  • the full sequence of human ⁇ -lactalbumin, including signal peptide, is provided as SEQ ID NO:36.
  • the sequence of mature human ⁇ -lactalbumin following cleavage of the signal peptide is provided as SEQ ID NO:35.
  • Table 3 - Human Lactoferrin sequences Protein Sequence SEQ ID NO Full length human ⁇ -lactalbumin 36 Mature human ⁇ -lactalbumin 35 SP1-human ⁇ -lactalbumin 37 SP2-human ⁇ -lactalbumin 38 SP3-human ⁇ -lactalbumin 39 SP4-human ⁇ -lactalbumin 40
  • a human ⁇ -lactalbumin of the present disclosure is a recombinant human ⁇ -lactalbumin.
  • a recombinant human ⁇ -lactalbumin of the disclosure is obtained from a mammalian, fungal, yeast, bacterial, or other cell.
  • a recombinant human ⁇ -lactalbumin of the disclosure is not obtained from a mammalian cell.
  • a recombinant human ⁇ -lactalbumin of the disclosure is obtained from a yeast cell.
  • the yeast cell may be, for example, a Arxula, Aspegillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodamaea, Komagataella, Leucosporidiella, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickerhamomyces, or Yarrowia cell.
  • the yeast cell is yeast cell is a Komagataella cell (e.g., Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris ) . Additional yeast cells suitable for recombinant protein production are recognized in the art and contemplated herein.
  • a recombinant human ⁇ -lactalbumin of the disclosure is obtained from a bacterial cell.
  • a human ⁇ -lactalbumin of the disclosure is isolated from a natural source.
  • human ⁇ -lactalbumin having at least one hybrid or complex N-glycan.
  • the human ⁇ -lactalbumin comprises a glycan comprising one or more of sialic acid, galactose, N-acetylgalactosamine, or fucose.
  • the human lactoferrin comprises a bi-antennary, tri-antennary, or tetra-antennary N-glycan.
  • human ⁇ -lactalbumin having one or more hybrid, complex, bi-antennary, tri-antennary, or tetra-antennary N-glycan may be useful in, for example, infant formula or other nutritional compositions or supplements.
  • compositions e.g., infant formula compositions
  • methods of the disclosure include, but are not limited to, secretory IgA (sIgA), human serum albumin, xanthine dehydrogenase, lactoperoxidase, butyrophilin, lactadherin, adiponectin, ⁇ -casein, ⁇ -casein, leptin, osteopontin, bile salt stimulated lipase (BSSL), and lysozyme. Any one or more of these human milk proteins may be included in compositions (e.g., infant formula) of the present disclosure. Any one or more of these human milk proteins may be excluded in certain embodiments.
  • secretory IgA secretory IgA
  • human serum albumin xanthine dehydrogenase
  • lactoperoxidase lactoperoxidase
  • butyrophilin lactadherin
  • lactadherin lactadherin
  • an N-acetylglucosaminyltransferase protein describes any polypeptide having N-acetylglucosaminyltransferase activity.
  • An N-acetylglucosaminyltransferase describes an enzyme that catalyzes the transfer of a monosaccharide from specific sugar nucleotide donors onto particular hydroxyl position of a monosaccharide in a growing glycan chain in one of two possible anomeric linkages (either ⁇ or ⁇ ).
  • N-acetylglucosaminyltransferase protein may be an N-acetylglucosaminyltransferase protein from any suitable organism.
  • the N-acetylglucosaminyltransferase protein is a eukaryotic N-acetylglucosaminyltransferase protein.
  • the N-acetylglucosaminyltransferase protein is a mammalian N-acetylglucosaminyltransferase protein.
  • the N-acetylglucosaminyltransferase protein is an N-acetylglucosaminyltransferase I protein (EC 2.4.1.101).
  • the systematic name of this enzyme class is Alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase.
  • Other names include: GnT-I, N-acetylglucosaminyltransferase I, and Uridine diphosphoacetylglucosamine-alpha-1,3-mannosylglycoprotein beta-1,2-N-acetylglucosaminyltransferase.
  • an N-acetylglucosaminyltransferase I protein of the present disclosure is Homo sapiens GnT-I, however a N-acetylglucosaminyltransferase I protein from any eukaryotic organism may be used as a part of the methods and composition of the disclosure..
  • the N-acetylglucosaminyltransferase protein is a ⁇ -1,2-N-acetylglucosaminyltransferase protein (EC 2.4.1.143).
  • the systematic name of this enzyme class is Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase.
  • Other names include: GnT-II, N-acetylglucosaminyltransferase II, and Uridine diphosphoacetylglucosamine-alpha-1,6-mannosylglycoprotein beta-1-2-N-acetylglucosaminyltransferase.
  • a ⁇ -1,2-N-acetylglucosaminyltransferase protein of the present disclosure is Rattus norvegicus GnT-II, however a ⁇ -1,2-N-acetylglucosaminyltransferase protein from any eukaryotic organism may be used as a part of the methods and composition of the disclosure.
  • an " ⁇ -1,3/6-Mannosidase protein” (or “alpha-1,3/6-Mannosidase protein”) describes any polypeptide having ⁇ -1,3/6-Mannosidase activity.
  • An ⁇ -1,3/6-Mannosidase describes an enzyme that catalyzes removal of two mannosyl residues from N-glycans. The systematic name of this enzyme class is Mannosyl-oligosaccharide 1,3-1,6-alpha-mannosidase. Other names include: Man-II and Mannosidase II.
  • an ⁇ -1,3/6-Mannosidase protein may be from any suitable organism.
  • the ⁇ -1,3/6-Mannosidase protein is a eukaryotic ⁇ -1,3/6-Mannosidase protein.
  • the ⁇ -1,3/6-Mannosidase protein is Drosophila melanogaster Man-II, however a ⁇ -1,3/6-Mannosidase protein from any eukaryotic organism may be used as a part of the methods and composition of the disclosure.
  • ⁇ -1,2-mannosidase protein (EC 3.2.1.130).
  • a " ⁇ -1,2-mannosidase protein” (or “alpha-1,2-mannosidase protein”) describes any polypeptide having ⁇ -1,2-mannosidase activity.
  • the systematic name of this enzyme class is Glycoprotein endo-alpha-1,2-mannosidase. Other names include: Endo-alpha-D-mannosidase and Man-I.
  • the ⁇ -1,2-mannosidase protein is a fungal Man-I.
  • the Man-I is a Trichoderma reesei Man-I.
  • Beta-1,4- galactosyltransferase ( ⁇ -1,4-galactosyltransferase)
  • ⁇ -1,4-galactosyltransferase protein (EC 2.4.1.38).
  • a " ⁇ -1,4-galactosyltransferase protein” (or “beta-1,4-galactosyltransferase protein") describes any polypeptide having ⁇ -1,4-galactosyltransferase activity.
  • the systematic name of this enzyme class is Beta-N-acetylglucosaminylglycopeptide beta-1,4-galactosyltransferase.
  • Glycoprotein 4-beta-galactosyltransferase UDP-galactose--glycoprotein galactosyltransferase
  • GalT Glycoprotein 4-beta-galactosyltransferase
  • UDP-galactose--glycoprotein galactosyltransferase UDP-galactose--glycoprotein galactosyltransferase
  • GalT a mammalian GalT.
  • the GalT is a Homo Sapiens GalT.
  • glycoproteins of the disclosure are N-linked glycoproteins.
  • N-linked glycoproteins contain an N-acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in the protein.
  • glycoproteins The predominant sugars found on glycoproteins are glucose, galactose, mannose, fucose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and sialic acid, e.g., N-acetyl-neuraminic acid (NANA).
  • the processing of the sugar groups occurs co-translationally in the lumen of the ER and continues in the Golgi apparatus for N-linked glycoproteins.
  • Certain aspects of the present disclosure include cells expressing one or more proteins from a nucleic acid molecule, where the protein is targeted to a desired subcellular location (e.g., an organelle such as the Golgi Apparatus).
  • a protein is targeted to a subcellular location by forming a fusion protein comprising a portion of the protein (e.g., a catalytic domain of an enzyme) and a cellular targeting signal peptide, e.g., a heterologous signal peptide (e.g., a signal peptide comprising SEQ ID NO:1, 2, 3, or 4) which is not normally ligated to or associated with the portion of the protein.
  • the fusion protein may be encoded by a polynucleotide encoding a cellular targeting signal peptide ligated in the same translational reading frame ("in-frame") to a nucleic acid fragment encoding a protein (e.g., enzyme), or catalytically active fragment thereof.
  • in-frame translational reading frame
  • the targeting signal peptide component of the fusion construct or protein may be derived from membrane-bound proteins of the ER or Golgi, retrieval signals, Type II membrane proteins, Type I membrane proteins, membrane spanning nucleotide sugar transporters, mannosidases, sialyltransferases, glucosidases, mannosyltransferases and phosphomannosyltransferases.
  • the targeting signal peptide is a Golgi Apparatus localization tag.
  • Example Golgi Apparatus localization tags include, but are not limited to, a transmembrane domain from Saccharomyces cerevisiae Kre2p, Saccharomyces cerevisiae Mnn2p, Saccharomyces cerevisiae Mnn9, Komagatella phaffii Bmt2, Komagatella phaffii Bmt3, or Komagatella phaffii Ktr2.
  • Vectors for transforming microorganisms in accordance with the present disclosure can be prepared by known techniques familiar to those skilled in the art in view of the disclosure herein.
  • a vector typically contains one or more genes, in which each gene codes for the expression of a desired product (the gene product) and is operably linked to one or more control sequences that regulate gene expression or target the gene product to a particular location in the recombinant cell.
  • Exogenous nucleic acid sequences including, for example, nucleic acid sequences encoding fusion proteins, nucleic acid sequences encoding wild-type or mutant proteins, may be introduced into many different host cells.
  • Nucleic acid sequences configured to facilitate a genetic mutation in a gene may also be introduced into various host cells, as described further herein. Suitable host cells are microbial hosts that can be found broadly within the fungal families.
  • Suitable host strains include but are not limited to fungal or yeast species, such as Arxula, Aspegillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Hansenula, Kluyveromyces, Komagataella, Leucosporidiella, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, and Yarrowia.
  • a host cell of the present disclosure is a Komagataella cell.
  • a host cell of the present disclosure is Komagataella phaffii. In some embodiments, a host cell of the present disclosure is Komagataella pastoris. In some embodiments, a host cell of the present disclosure is Komagataella pseudopastoris.
  • Microbial expression systems and expression vectors are well known to those skilled in the art. Any such expression vector could be used to introduce the instant genes and nucleic acid sequences into an organism.
  • the nucleic acid sequences may be introduced into appropriate microorganisms via transformation techniques. For example, a nucleic acid sequence can be cloned in a suitable plasmid, and a parent cell can be transformed with the resulting plasmid.
  • the plasmid is not particularly limited so long as it renders a desired nucleic acid sequence inheritable to the microorganism's progeny.
  • Vectors or cassettes useful for the transformation of suitable host cells are recognized in the art.
  • the vector or cassette contains a gene, sequences directing transcription and translation of a relevant gene including the promoter, a selectable marker, and sequences allowing autonomous replication or chromosomal integration.
  • Suitable vectors comprise a region 5' of the gene harboring the promoter and other transcriptional initiation controls and a region 3' of the DNA fragment which controls transcriptional termination.
  • Promoters, cDNAs, and 3'UTRs, as well as other elements of the vectors can be generated through cloning techniques using fragments isolated from native sources ( Green & Sambrook, Molecular Cloning: A Laboratory Manual, (4th ed., 2012 ); U.S. Pat. No. 4,683,202 ; incorporated by reference). Alternatively, elements can be generated synthetically using known methods ( Gene 164:49-53 (1995 )).
  • Vectors for transforming microorganisms in accordance with the present disclosure can be prepared by known techniques familiar to those skilled in the art in view of the disclosure herein.
  • a vector typically contains one or more genes, in which each gene codes for the expression of a desired product (the gene product) and is operably linked to one or more control sequences (e.g., promoter sequences, signal peptide sequences) that regulate gene expression or target the gene product to a particular location in the recombinant cell.
  • control sequences e.g., promoter sequences, signal peptide sequences
  • Control sequences are nucleic acid sequences that regulate the expression of a coding sequence or direct a gene product to a particular location in or outside a cell.
  • Control sequences that regulate expression include, for example, promoters that regulate transcription of a coding sequence and terminators that terminate transcription of a coding sequence.
  • Another control sequence is a 3' untranslated sequence located at the end of a coding sequence that encodes a polyadenylation signal.
  • Control sequences that direct gene products to particular locations include those that encode signal peptides, which direct the protein to which they are attached to a particular location inside or outside the cell.
  • an example vector design for expression of a gene in a microbe contains a coding sequence for a desired gene product (for example, a selectable marker, an enzyme, a fusion protein, etc.) in operable linkage with a promoter active in yeast.
  • a desired gene product for example, a selectable marker, an enzyme, a fusion protein, etc.
  • the coding sequence can be transformed into the cells such that it becomes operably linked to an endogenous promoter at the point of vector integration.
  • Example promoters contemplated herein include, but are not limited to, the AOX1, GAP, TEF1, TPI1, DAS1, DAS2, CAT1, and FMD promoters.
  • the promoter used to express a gene can be the promoter naturally linked to that gene or a different promoter.
  • a promoter can generally be characterized as constitutive or inducible. Constitutive promoters are generally active or function to drive expression at all times (or at certain times in the cell life cycle) at the same level. Inducible promoters, conversely, are active (or rendered inactive) or are significantly up- or down-regulated only in response to a stimulus. Both types of promoters find application in the disclosed methods. Useful inducible promoters include those that mediate transcription of an operably linked gene in response to a stimulus, such as an exogenously provided small molecule, temperature (heat or cold), lack of nitrogen in culture media, etc. Suitable promoters can activate transcription of an essentially silent gene or upregulate transcription of an operably linked gene that is transcribed at a low level.
  • termination region control sequence may be native to the transcriptional initiation region (the promoter), may be native to the DNA sequence of interest, or may be obtainable from another source (See, e.g., Chen & Orozco, Nucleic Acids Research 16:8411 (1988 )).
  • the full nucleotide sequence of a promoter is not necessary to drive transcription, and sequences shorter than the promoter's full nucleotide sequence can drive transcription of an operably-linked gene.
  • the minimal portion of a promoter termed the core promoter, includes a transcription start site, a binding site for a RNA polymerase, and a binding site for a transcription factor.
  • a promoter may be linked to a target by introducing the promoter and the target into a nucleic acid molecule, for example, a vector.
  • a vector may be introduced into a cell, thereby expressing the promoter and the target.
  • a promoter is linked to a target by introducing a promoter into DNA of a cell, for example, via homologous recombination, thereby integrating the promoter into the genome of the cell.
  • a gene typically includes a promoter, a coding sequence, and termination control sequences.
  • a gene When assembled by recombinant DNA technology, a gene may be termed an expression cassette and may be flanked by restriction sites for convenient insertion into a vector that is used to introduce the recombinant gene into a host cell.
  • the expression cassette can be flanked by DNA sequences from the genome or other nucleic acid target to facilitate stable integration of the expression cassette into the genome by homologous recombination.
  • the vector and its expression cassette may remain unintegrated (e.g., an episome), in which case, the vector typically includes an origin of replication, which is capable of providing for replication of the vector DNA.
  • a common gene present on a vector is a gene that codes for a protein, the expression of which allows the recombinant cell containing the protein to be differentiated from cells that do not express the protein.
  • a gene, and its corresponding gene product is called a selectable marker or selection marker. Any of a wide variety of selectable markers can be employed in a transgene construct useful for transforming the organisms covered in the disclosed embodiments.
  • transgenic messenger RNA mRNA
  • codon usage in the transgene is not optimized, available tRNA pools may not be sufficient to allow for efficient translation of the transgenic mRNA resulting in ribosomal stalling and termination and possible instability of the transgenic mRNA.
  • a coding sequence of the present disclosure can be codon optimized for a particular host cell by replacing one or more rare codons with one or more codons more frequently found in the host cell.
  • a rare codon in a host cell describes a codon that is found in less than 5%, less than 10%, or less than 20% of coding sequences in the host cell. Rare codons can be identified using methods known to those of skill in the art.
  • aspects of the disclosure comprise transformation of a microorganism with a nucleic acid sequence comprising a gene that encodes a protein.
  • the gene may be native to the cell or from a different species.
  • the gene may be derived from a different species yet modified (e.g., codon optimized) for optimal expression in the microorganism.
  • the gene is inheritable to the progeny of a transformed cell.
  • the gene is inheritable because it resides on a plasmid.
  • the gene is inheritable because it is integrated into the genome of the transformed cell.
  • aspects of the disclosure may comprise transformation of a microorganism with a nucleic acid sequence configured to generate a mutation in a gene of the microorganism.
  • aspects of the disclosure may comprise transformation of the microorganism with a nucleic acid sequence comprising sequences upstream and downstream of a gene (e.g., an OCH1 gene), thereby facilitating reduced expression or deletion of the gene via homologous recombination.
  • a gene e.g., an OCH1 gene
  • Various methods for generating mutations (including deletions or knockout mutations, as well as mutations which reduce expression of a gene) in genes of a microorganism are recognized in the art and envisioned herein.
  • a microorganism having a deletion or knockout mutation of a gene does not product a functional copy of the protein.
  • a recombinant yeast cell of the disclosure may comprise a deletion of an endogenous OCH1 gene, such that the recombinant yeast cell does not express an endogenous, functional OCH1 protein.
  • a microorganism having a reduced expression of a gene or protein produces a functional copy of the protein, but at a reduced amount compared with a wild-type (i.e., a non-recombinant or non-genetically modified) microorganism of the same species.
  • Methods for reducing expression of a protein are recognized in the art and include, for example, replacement of an endogenous promoter and/or modification of one or more regulatory elements.
  • Cells can be transformed by any suitable technique including, e.g., biolistics, electroporation, glass bead transformation, and silicon carbide whisker transformation. Any convenient technique for introducing a transgene into a microorganism can be employed in the embodiments disclosed herein.
  • an exemplary vector design for expression of a gene in a microorganism contains a gene encoding an enzyme in operable linkage with a promoter active in the microorganism.
  • the gene can be transformed into the cells such that it becomes operably linked to a native promoter at the point of vector integration.
  • the vector can also contain a second gene that encodes a protein.
  • one or both gene(s) is/are followed by a 3' untranslated sequence containing a polyadenylation signal.
  • Expression cassettes encoding the two genes can be physically linked in the vector or on separate vectors. Co-transformation of microbes can also be used, in which distinct vector molecules are simultaneously used to transform cells ( Protist 155:381-93 (2004 )). The transformed cells can be optionally selected based upon the ability to grow in the presence of the antibiotic or other selectable marker under conditions in which cells lacking the resistance cassette would not grow.
  • aspects of the disclosure comprise genetically engineered cells (also “engineered cells” or “recombinant cells”) and methods for making and using such cells.
  • recombinant cells comprising one or more exogenous nucleic acid sequences.
  • methods for generating such recombinant cells comprising introducing the one or more exogenous nucleic acid sequences into a host cell.
  • methods for collecting one or more products e.g., a mammalian protein
  • the recombinant cell is a prokaryotic cell, such as a bacterial cell.
  • the recombinant cell is a eukaryotic cell, such as a mammalian cell, a yeast cell, a filamentous fungi cell, a protist cell, an algae cell, an avian cell, a plant cell, or an insect cell.
  • the cell is a yeast cell.
  • a recombinant cell of the disclosure may be selected from the group consisting of algae, bacteria, molds, fungi, plants, and yeasts.
  • a recombinant cell of the disclosure is a bacterial cell (e.g. E.coli ), a fungal cell, or a yeast cell.
  • a recombinant cell of the disclosure is a recombinant fungal cell.
  • a recombinant fungal cell may be any suitable fungal cell recognized in the art.
  • the fungal cell is an Arxula, Aspegillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodamaea, Komagataella, Leucosporidiella, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickerhamomyces, or Yarrowia cell.
  • the fungal cell is Arxula adeninivorans, Aspergillus niger, Aspergillus orzyae, Aspergillus terreus, Aurantiochytrium limacinum, Candida utilis, Claviceps purpurea, Cryptococcus albidus, Cryptococcus curvatus, Cryptococcus ramirezgomezianus, Cryptococcus terreus, Cryptococcus wieringae, Cunninghamella echinulata, Cunninghamella japonica, Geotrichum fermentans, Hansenula polymorpha, Kluyveromyces lactis, Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris, Kluyveromyces marxianus, Kodamaea ohmeri, Leucosporidiella creatinivora, Lipomyces lipofer, Lipomyces starkeyi, Lipomyces tetrasporus
  • the fungal cell is a yeast cell.
  • the yeast cell is a Komagataella cell.
  • the yeast cell is Kluyveromyces phaffii, Komagataella pastoris, or Komagataella pseudopastoris.
  • the yeast cell is Kluyveromyces phaffii.
  • an engineered cell of the present disclosure is a yeast cell comprising one or more modifications for improving generation of N-glycans including human-like N-glycans. Examples of such cells and modifications are described in, for example, US patent 9,617,550 , incorporated herein by reference in its entirety.
  • Certain embodiments of the disclosure are directed to the use of gene editing techniques to generate a knockout or other mutation in a gene in a population of cells.
  • Various methods and systems for gene editing are known in the art and include, for example, zinc finger nuclease (ZFN)-based gene editing, transcription activator-like effector nuclease (TALEN)-based gene editing, and CRISPR/Cas-based gene editing.
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • CRISPR/Cas-based gene editing are recognized in the art and contemplated herein.
  • methods of the present disclosure comprise CRISPR/Cas-based gene editing, which comprises the use of components of a CRISPR system, for example a guide RNA (gRNA) and a Cas nuclease.
  • gRNA guide RNA
  • Cas nuclease for example a guide RNA (gRNA)
  • gRNA guide RNA
  • CRISPR system refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a "direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a "spacer” in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus.
  • a tracr trans-activating CRISPR
  • tracr-mate sequence encompassing a "direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system
  • guide sequence also referred to as a "spacer” in the context of an endogenous CRISPR
  • the CRISPR/Cas nuclease or CRISPR/Cas nuclease system can include a noncoding RNA molecule (guide) RNA, which sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9), with nuclease functionality (e.g., two nuclease domains).
  • a CRISPR system can derive from a type I, type II, or type III CRISPR system, e.g., derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.
  • a Cas nuclease and gRNA are introduced into the cell.
  • a Cas nuclease and a gRNA can be introduced into the cell indirectly via introduction of one or more nucleic acids (e.g., vectors) encoding for the Cas nuclease and/or the gRNA.
  • a Cas nuclease and a gRNA can be introduced into the cell directly by introduction of a Cas nuclease protein and a gRNA molecule.
  • target sites at the 5' end of the gRNA target the Cas nuclease to the target site, e.g., the gene, using complementary base pairing.
  • the target site may be selected based on its location immediately 5' of a protospacer adjacent motif (PAM) sequence, such as typically NGG, or NAG.
  • PAM protospacer adjacent motif
  • the gRNA may be targeted to the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence.
  • a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence.
  • target sequence generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.
  • the CRISPR system can induce double stranded breaks (DSBs) at the target site, followed by disruptions as discussed herein.
  • Cas9 variants deemed “nickases,” are used to nick a single strand at the target site. Paired nickases can be used, e.g., to improve specificity, each directed by a pair of different gRNAs targeting sequences such that upon introduction of the nicks simultaneously, a 5' overhang is introduced.
  • catalytically inactive Cas9 is fused to a heterologous effector domain such as a transcriptional repressor or activator, to affect gene expression.
  • the target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides.
  • the target sequence may be located in the nucleus or cytoplasm of the cell, such as within an organelle of the cell.
  • a sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an "editing template” or "editing polynucleotide” or “editing sequence”.
  • an exogenous template polynucleotide may be referred to as an editing template.
  • the recombination is homologous recombination.
  • the CRISPR complex (comprising the guide sequence hybridized to the target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence.
  • the tracr sequence which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g.
  • tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of the CRISPR complex, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned.
  • One or more vectors driving expression of one or more elements of a CRISPR system can be introduced into a cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites.
  • Components can also be delivered to cells as proteins and/or RNA.
  • a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors.
  • two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector.
  • the vector may comprise one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a "cloning site").
  • a restriction endonuclease recognition sequence also referred to as a "cloning site”
  • one or more insertion sites are located upstream and/or downstream of one or more sequence elements of one or more vectors.
  • a vector may comprise a regulatory element operably linked to an enzyme-coding sequence encoding a Cas protein (also "Cas nuclease”).
  • Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas12a (Cpf1), Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof,
  • the Cas nuclease can be Cas9 (e.g., from S. pyogenes or S. pneumonia).
  • the Cas nuclease can be Cas12a.
  • the Cas nuclease can direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence.
  • the vector can encode a Cas nuclease that is mutated with respect to a corresponding wild-type enzyme such that the mutated Cas nuclease lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence.
  • a Cas9 nickase may be used in combination with guide sequence(s), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ or HDR.
  • guide sequence(s) e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ or HDR.
  • an enzyme coding sequence encoding the CRISPR enzyme is codon optimized for expression in particular cells, such as yeast cells.
  • a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence.
  • the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm is or is more than 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), Clustal W, Clustal X, BLAST, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • any suitable algorithm for aligning sequences include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), Clustal W, Clustal X, BLAST, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and
  • the Cas nuclease may be part of a fusion protein comprising one or more heterologous protein domains.
  • a Cas nuclease fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a Cas nuclease, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.
  • Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • reporter genes include, but are not limited to, glutathione-5- transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP).
  • GST glutathione-5- transferase
  • HRP horseradish peroxidase
  • CAT chloramphenicol acetyltransferase
  • beta galactosidase beta-glucuronidase
  • a Cas nuclease may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may form part of a fusion protein comprising a Cas nuclease are described in US 20110059502 , incorporated herein by reference.
  • Example 1 Novel signal peptides increase extracellular protein levels
  • DNA encoding SEQ ID NO:1 (“SP1”), SEQ ID NO:2 (“SP2”), and SEQ ID NO:4 (“SP4") was cloned in-frame in the 5' end of the DNA coding for a protein of interest (POI), i.e., Pichia pastoris codon-optimized human lactoferrin, resulting in the substitution of the pre-pro-MF ⁇ from Saccharomyces cerevisiae. This is the most widely used signal peptide in yeast and served as the control. Single copies of the resulting sequences and the control were integrated into the AOX1 locus via double-crossover. Multiple colonies of each transformation plate were cultivated in 96-deep well plates.
  • POI protein of interest
  • Oligonucleotides and gBlocks were ordered from Integrated DNA Technologies (San Diego, CA, USA) and are described in Table 5.
  • NEBuilder@ HiFi DNA Assembly Master Mix, OneTaq ® Quickload ® DNA polymerase, and Escherichia coli DH5 ⁇ cells were from New England Biolabs. All polymerase chain reaction (PCR)-amplified sequences were confirmed via sequencing at or Genewiz.
  • Transformation of linear dsDNA for integration was performed using the method described by Madden, Tolstorukov, & Cregg (2014) Fungi, Volume 1, Fungal Biology .
  • Total yeast genomic DNA extraction was performed using the kit Easy DNA from Invitrogen (ThermoFisher, Applied Biosystems TM , PrepSEQ TM 1-2-3 Nucleic Acid Extraction Kit, Catalog number: 4452222).
  • the resulting plasmids are summarized in Table 6.
  • the leader peptide sequences from the Pichia pastoris endogenous proteins Ost1 and Pst1 were determined using SignalP-5.0 bioinformatic software, publicly available from the Center Biological Sequence Analysis (CBS).
  • CBS Center Biological Sequence Analysis
  • the pro region of Epx1 was described by Heiss et al. (2015) Microbiology, 161(7 ).
  • Plasmid P1 containing the gene encoding human lactoferrin without its native secretion peptide fused in-frame with the pre-pro-leader peptide of the mating factor-alpha from Saccharomyces cerevisiae was synthesized by Genscript.
  • the human lactoferrin gene was codon-optimized for expression in Pichia pastoris.
  • primers PMR1 SEQ ID NO:16
  • PMR2 SEQ ID NO:17
  • the backbone containing human lactoferrin, yeast HIS4 auxotrophic marker, and Escherichia coli antibiotic resistance and origin of replication was obtained via polymerase chain reaction (PCR) of P1 plasmid using primers PMR3 (SEQ ID NO:18) and PMR4 (SEQ ID NO: 19).
  • PCR polymerase chain reaction
  • primers PMR5 SEQ ID NO:20
  • PMR6 SEQ ID NO:21
  • gBLOCK1 SEQ ID NO: 15
  • the backbone containing human lactoferrin, yeast HIS4 auxotrophic marker, and Escherichia coli antibiotic resistance and origin of replication was obtained via PCR of P1 plasmid with primers PMR7 (SEQ ID NO:22) and PMR8 (SEQ ID NO:23).
  • the two resulting fragments were assembled using NEBuilder@ HiFi DNA Assembly Master Mix following manufacturer instructions
  • primers PMR9 SEQ ID NO:24
  • PMR10 SEQ ID NO:25
  • the backbone containing human lactoferrin, yeast HIS4 auxotrophic marker, and Escherichia coli antibiotic resistance and origin of replication was obtained via PCR of P1 plasmid with primers PMR11 (SEQ ID NO:26) and PMR12 (SEQ ID NO:27).
  • the two resulting fragments were assembled using NEBuilder@ HiFi DNA Assembly Master Mix following manufacturer instructions
  • primers PMR13 (SEQ ID NO:28) and PMR14 (SEQ ID NO:29) were used for amplification using the gBLOCK1 (SEQ ID NO: 15) as a template.
  • the backbone containing human lactoferrin, yeast HIS4 auxotrophic marker, and Escherichia coli antibiotic resistance and origin of replication was obtained via PCR of P1 plasmid with primers PMR15 (SEQ ID NO:30) and PMR16 (SEQ ID NO:31).
  • the two resulting fragments were assembled using NEBuilder@ HiFi DNA Assembly Master Mix following manufacturer instructions
  • Assembly mixtures were transformed into Escherichia coli DH5 ⁇ cells as directed by the manufacturer and plated into Luria Broth (LB)-agar plates containing 100 ⁇ g/mL of ampicillin. Positive clones were selected via colony polymerase chain reaction (PCR) and inoculated overnight in 5 mL of liquid Luria Broth media supplemented with 100 ⁇ g/mL of ampicillin. Plasmids from Escherichia coli cells were isolated using GeneJET plasmid miniprep kit (ThermoFisher ® , Catalog number K0502). Proper assembly was confirmed via Sanger DNA sequencing.
  • Linear dsDNA fragment for integration into yeast was obtained using Q5 High-Fidelity DNA polymerase using primers PMR17 (SEQ ID NO:32) and PMR18 (SEQ ID NO:33) and plasmids P1, P2, P3, P4, or P5 as a template.
  • Electrocompetent Pichia pastoris cells were transformed as described by Madden, Tolstorukov, & Cregg (2014) Fungi, Volume 1, Fungal Biology . Cells were spread on MD plates (1.34% yeast nitrogen base, 4 ⁇ 10 -5 % biotin, 2% dextrose, 20% agar), which allows for selection of his4 + cells, and incubated at 30°C for seventy-two hours.

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